A cluster of Antigen 5 family members expressed in the salivary glands of the African malaria vector Anopheles gambiae.

Salivary glands of disease vectors are an interesting topic in molecular entomological studies not only for the crucial role they play in the transmission of different pathogens but also for the production of a large number of secreted factors whose anti-haemostatic, anti-inflammatory and immuno-modulatory activity are of essential importance for blood-feeding. Moreover, as recently shown for the sandfly vector Phlebotomus papatasi and the parasite Leishmania major, salivary antigens may prove useful vaccine components (Valenzuela JG et al, 2001 J Exp Med, 194: 331-342). For these reasons, using a cloning strategy suitable for the trapping of salivary genes encoding secreted factors, we identified, in the last few years, more than twenty cDNAs whose expression is specific or enriched in the Anopheles gambiae salivary glands. One of the cloned cDNA fragments was a member of the widespread family of insect proteins known as Antigen 5 (Ag5) and, because of its similarity to venom allergens from ants and wasps, we named the corresponding gene gVAG (gambiae Venom AllerGen) (Arcà B et al, 1999 Proc Natl Acad Sci USA, 96: 1516-1521). The sequence of the 933 bp full-length cDNA was reconstructed by the 5’ RACE technique (Rapid Amplification of cDNA Ends) and has the potential to encode a protein of 260 amino acids. Three members of the Ag5 family were also identified by Ribeiro and collaborators from an A. gambiae salivary gland cDNA library and named respectively Ag5-related 1 (A5r1), Ag5-related 2 (A5r2) and Ag5-related 3 (A5r3) (Francischetti IM et al, 2002, J Exp Biol, 205: 2429-2451). Interestingly, A5r1 and gVAG seem to derive from two alternative spliced transcripts of the A5r1/gVAG gene. Insect Ag5-related proteins belong to the CAP family that also includes mammalian cysteine-rich secretory proteins (CRISPs) and plant pathogenesis-related proteins (PR proteins). Members of this gene family appear to encode secreted factors but their function has not yet been clarified. Ag5 family members are expressed in the saliva of several blood-sucking Diptera (such as mosquitoes, sand flies and tsetse) and Hemiptera (Rhodnius prolixus) and this observation suggests that they are likely to play some essential role associated to blood feeding. Using the gVAG polipeptide as a query we searched the A. gambiae genome and we found that it contains 20 members of the Ag5 family. Notably, chromosome arm 2L contains a cluster of five Ag5-related genes that includes the previously mentioned A5r1 and A5r2, whereas A5r3 is located on the X chromosome. Since several salivary genes are clustered together we decided to verify the tissue and developmental pattern of expression of the other members of the Ag5 cluster, that we named A5r4, A5r5 and A5r6. Moreover, we also analyzed two additional family members located on the same chromosome respectively ~15 Mb total RNA extracted from different tissues/developmental stages (embryos, larvae, pupae, female salivary glands, female carcasses and adult males) to amplify the corresponding mRNAs by RT-PCR. upstream (A5r8) and 0.4 Mb downstream (A5r7). To this purpose we used gene-specific primers and All the genes of the cluster were found expressed, even if not exclusively, in the female salivary glands with the only exception of A5r5 that was expressed only in adult males. On the contrary, A5r7 and A5r8, located outside of the cluster on the same chromosome arm, were not expressed in female salivary glands. Notably, also these last two genes, as A5r1, show an alternative splicing with a short intron that is retained in adult males and carcasses (i.e. adult females with salivary glands removed) and spliced in embryos and larvae; in all these cases the unspliced transcripts have the potential to encode truncated forms of the putative respective proteins. The significance of this alternative splicing is not known but the expression pattern of several Antigen 5 family members in the A. gambiae female salivary glands indicates that they likely play some important function in connection to blood-feeding. Expression of recombinant forms of these proteins followed by functional studies may allow in the next future a better understanding of their physiological role.